Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and ; radiation

Ku protein is a heterodimer (Ku70 and Ku86) known to play an important role in V(D)J recombination, apoptosis, telomere fusion, and double-strand break repair. Its role in double-strand breaks is relevant to cancer therapy because lack of Ku86 causes one of the most radiationresponsive phenotypes (hamster cells, XRS5). Although it is known that the heterodimer is necessary for the various functions of this protein, the impact of targeting Ku in human cancer cells has not been shown due to lack of appropriate approaches. It is also not known whether complete knock-out of Ku protein is required to enhance the sensitivity of human cells to ; radiation as Ku protein is much more abundant in human cells than in hamster cells. In the current article, we have investigated the direct effect of Ku70 depletion in human cervical epithelioid (HeLa) and colon carcinoma (HCT116) cells. We specifically targeted Ku70 mRNA by use of small interfering RNA (siRNA). Of the five Ku70 siRNA synthesized, three inhibited the expression of Ku70 by up to 70% in HeLa cells. We have tested the effect of chemically synthesized siRNAs for target sequence 5 (CS #5) on the response of HeLa cells 72 hours after transfection to ; radiation and etoposide, as this showed the maximum inhibition of Ku70 expression. Ku70 siRNA induced a decrease in the surviving fraction of irradiated HeLa cells by severalfold. Similar sensitizing effects were observed for etoposide, a topoisomerase II inhibitor. Studies with HCT116 cells using the same Ku70 siRNA (CS #5) showed a direct correlation between expression of Ku70 and sensitization to radiation and etoposide treatments. [Mol Cancer Ther 2005;4(4):529–36] Introduction It has been a long-standing interest to target proteins responsible for the resistance of cancer cells to g radiation and chemotherapeutic agents (1–4). Most recent molecular biological studies have focused on the signaling transduction mechanisms responsible for cellular proliferation and growth (4–7). It is quite possible that targeting signaling pathways mediated by phosphorylation, kinases, growth receptors, and oncogenes may yield clinically relevant approaches to specifically kill cancer cells; provided that only cancer cells and not the normal cells rely on these pathways for survival. There are numerous preclinical evidences that these inhibitors can potentiate the antitumor effects of many cytotoxic agents (8). However, the efficacy of these inhibitors to enhance the effects of cytotoxic agents in clinical studies in humans is not clearly established (8). Baker et al. (9) have shown that overexpression of thioredoxin, which plays some role in the growth of several cancer cells, is effective in reducing the sensitivity of mammalian cells to certain chemotherapeutic agents. However, we have recently shown that inhibition of the oxidative pentose phosphate cycle, which supplies NADPH required for the function of thioredoxin and other proteins, did not sensitize the noncancerous Chinese hamster ovary cells to etoposide, and only slightly sensitized these cells to g radiation (10). These results have indicated that oxidative pentose phosphate cycle–mediated redox signaling might play only a minor role in cellular response to DNA-damaging agents. The most relevant downstream signaling pathways that could play a significant role in sensitizing the cancer cells to DNA-damaging agents are those involved in apoptosis (11–13). We have previously shown that DNA is an important target in radiation-induced apoptosis, suggesting that both mitotic and apoptotic cell death are caused by DNA lesions after irradiation (14). Ku protein plays a major role in the repair of DNA double-strand breaks caused by g radiation and some chemotherapeutic agents (15–19). A key system for the repair of DNA double-strand breaks is nonhomologous end-joining (20, 21). The role of Ku is to bind to DNA ends, thus facilitating the coordination of other DNA repair proteins (20, 21). Biochemical and genetic studies using mutant rodent cell lines sensitive to ionizing radiation have identified at least four genes, XRCC4, XRCC5, XRCC6, and XRCC7, that are required for nonhomologous end-joining (20, 21). XRCC4 encodes a 38 kDa nuclear phosphoprotein Received 5/21/04; revised 1/20/05; accepted 2/21/05. Grant support: National Cancer Institute research grant CA 92108. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Iraimoudi S. Ayene, Department of Radiation Oncology, School of Medicine, University of Pennsylvania, 195 John Morgan Building, Philadelphia, PA 19104-6072. Phone: 215-898-9507; Fax: 215-898-0090. E-mail: [email protected] Copyright C 2005 American Association for Cancer Research. 3 J.E. Biaglow et al., personal communication. 529 Mol Cancer Ther 2005;4(4). April 2005 on May 11, 2017. © 2005 American Association for Cancer Research. mct.aacrjournals.org Downloaded from that binds strongly to DNA-ligase IV (21). XRCC7 encodes a 460 kDa catalytic subunit (DNA-PKcs) of DNA-PK (20, 21). XRCC5 and XRCC6 encode 86 and 70 kDa subunits, respectively, of Ku autoantigen, a DNA end-binding protein and regulatory subunit of DNA-PK (20, 21). The protein kinase activity of DNA-PK is believed to be stimulated by its association with DNA-end-bound Ku (20, 21). Ku heterodimer binds to the ends of DNA in a non–sequencedependent manner (20, 21). The DNA-binding activity of Ku requires reduced sulfhydryl groups in cell-free systems (22). Ku heterodimer contains 14 cysteine residues and 5 of these are located in Ku70, which is in contact with the DNA binding site (22). In a recent report, we have shown that Ku protein function can be greatly decreased in glucose-6phosphate dehydrogenase–deficient Chinese hamster ovary cells by Ku protein thiol oxidation (23). However, this biochemical approach is effective only in glucose-6-phosphate dehydrogenase–deficient cells. In this study, we used a novel molecular approach to specifically target Ku70 mRNA. We show that this approach inhibits the expression of Ku70 protein and causes an increased response of cancer cells to g radiation and topoisomerase II inhibitor. Materials andMethods Cells and GrowthMedium HeLa and HCT116 cancer cells were obtained from the American Type Culture Collection (Manassas, VA). These cells were grown in McCoy 5A medium with 10% FCS and 20 mmol/L HEPES at 37jC in a humidified 5% CO2 incubator. Ku70 Small Interfering RNA Synthesis In vitro –Transcribed siRNA. The nucleotide sequence of human Ku gene (accession #BC010034) was retrieved from the National Center for Biotechnology Information. Twenty-one-mer nucleotides with starting amino acid nucleotides for several target sequences were selected using Ambion software. To produce in vitro –transcribed small interfering RNA (siRNA) against Ku70, we used the pSilencer siRNA construction kit according to the manufacturer’s recommendations (Ambion, Austin, TX). The sense and antisense for two target sequences Ku70-3 and Ku70-4 for in vitro –transcribed siRNA are shown below: Ku70-3 Sense 5V-AATTCAGGTGACTCCTCCAGG CCTGTCTC-3V Antisense 5V-AACCTGGAGGAGTCACCTGAA CCTGTCTC-3V